Combination of thienamycin-type antibiotics with dipeptidase inhibitors

ABSTRACT

A novel antibacterial drug combination is provided, one component being a 2-substituted penem, and the other component is a dipeptidase (E.C.3.4.13.11) inhibitor. The dual-component combination is formulated so that 1 to 3 parts by weight of the penem are employed for 30 to 1 parts by weight of the inhibitor compound.

RELATIONSHIP TO PRIOR APPLICATIONS

This is a continuation of application Ser. No. 394,311, filed July 7, 1982, now abandoned, which application is a continuation-in-part of U.S. Ser. No. 340,152, filed Jan. 18, 1982 now abandoned, which was a continuation-in-part of U.S. Ser. No. 291,711, filed Aug. 10, 1981, now U.S. Pat. No. 4,539,208 which was a continuation-in-part of U.S. Ser. No. 187,929, filed Sept. 17, 1980 now abandoned, which was a continuation-in-part of U.S. Ser. No. 050,232, filed June 22, 1979, now abandoned, which was in turn a continuation-in-part of U.S. Ser. No. 927,213, filed July 24, 1978, now abandoned.

The compounds 2-substituted penems are those having the following structure (described as the free acid, but also including easily removable or pharmaceutically acceptable salt or ester groups): ##STR1## wherein R is --R' or --SR' wherein R' is alkyl (including cycloalkyl) of 1-6 carbon atoms, aryl such as phenyl or phenylalkyl having 7-12 carbon atoms, heterocyclyl and heterocyclylalkyl wherein alkyl has 1-3 carbon atoms and the heterocycle has 1-4 hetero atoms selected from O, N, and S; such as 1-methyl-1,2,3,4-tetrazolyl, furyl, thienyl, pyridyl, thiazolyl, and similar examples. Alkyl or aryl as used in these definitions can also include substituted alkyl and substituted aryl, the substituents being alkoxy, alkyl carboxy, and others.

These penems as well as others are described by R. B. Woodward in "Recent Advances in the Chemistry of β-Lactam Antibiotics", J. Elks (Ed), The Chemical Society, London, 1977, p. 167; R. B. Woodward, Abstracts of Uppsala University 500 Years Symposium on Current Topics in Drug Research, Uppsala, Sweden, Oct. 19-21, 1977, Acta. Pharm. Suecica, Vol. 14, Supplement, p. 23, and U.S. Pat. Nos. 4,070,477, issued Jan. 24, 1978 and 4,260,618, issued Apr. 7, 1981, and in British patent application Nos. 2,013,674 published Aug. 15, 1979 and 2,042,520 published Sept. 24, 1980. All penems within these publications and patents are incorporated within the scope of this invention.

The particularly preferred group of penems for use in this invention as those in which R is SR', R' being alkyl of 1-6 carbon atoms, especially ethyl, or R' is pyridyl or phenyl.

These compounds have a high level of antibacterial activity, but are subject to extensive metabolism by mammalian species.

The kidney was identified as the primary site of metabolism, and an enzyme was purified from renal extracts which catalyzed the inactivation of thienamycin by hydrolysis of the β-lactam. By such criteria as cytological localization, substrate specificity and suceptibility to enzyme inhibitors, this enzyme is very similar if not identical to a widely studied renal dipeptidase (E.C.3.4.13.11), also referred to in the literature as "dehydropeptidase-I".

We have now found that these penems can be combined with a class of chemical compounds which are dipeptidase inhibitors, and which selectively inhibit the metabolism of the dipeptidase (E.C.3.4.13.11). These chemical compounds are Z-2-acylamino-3-monosubstituted propenoates having the following formula ##STR2## wherein R² and R³ are hydrocarbon radicals in the range respectively of 3-10 and 1-15 carbon atoms. In either of these hydrocarbon radicals R² and R³, up to 6 hydrogens may be replaced by halogens, or a non-terminal methylene may be replaced by oxygen or sulfur, including oxidized forms of the latter.

A terminal hydrogen in R³ can also be replaced by a hydroxyl or thiol group, which may be acylated, such as with an alkanoyl acid of 1-8 carbon atoms, or carbamoylated, including alkyl and dialkyl carbamate derivatives; or the hydrogen can be replaced by an amino group, which may be derivatized as in an acylamino, ureido, amidino, guanidino, or alkyl or substituted alkyl amino group, including quaternary nitrogen groupings; or, alternatively, there may be replacement by acid groups such as carboxylic, phosphonic or sulfonic acid groups or esters or amides thereof, as well as cyano; or combinations thereof, such as a terminal amino acid grouping.

R² is preferably a branched alkyl or cycloalkyl radical (C₃₋₁₀), with a limitation that the carbon adjacent to the carbonyl cannot be tertiary. R² cannot be phenyl or straight chain loweralkyl of 1-4 carbon atoms, where R³ is straight chain lower alkyl of 1-4 carbon atoms. R¹ is hydrogen, lower-alkyl (C₁₋₆) or dialkylaminoalkyl (e.g., --CH₂ CH₂ N(C₂ H₅)₂, --CH₂ CH(CH₃)N(CH₃)₂.

Some of the compounds with formula II above have asymmetric forms. Racemic Z-2-(2,2-dimethylcyclopropanecarboxamido)-2-octenoic acid has been resolved. The activity resides in the dextrorotatory isomer, which has the S-configuration.

Within the definition of R², the following sub-groups are included:

    --R.sup.4                                                  II A

wherein R⁴ is a straight, branched, or cyclic hydrocarbon radical of 3-10 carbon atoms which may be substituted as specified above in the definition of R² ;

    --R.sup.5 R.sup.6                                          II B

wherein R⁵ is cycloalkyl of 3-6 carbon atoms and R⁶ is either 1 or 2 alkyl substituents which may be joined to form another ring on the cycloalkyl group, or R⁵ and R⁶ may be substituted as specified above in the definition of R² ;

    --R.sup.7 R.sup.8                                          II C

wherein R⁷ is an alkylene group of 1-3 carbon atoms and R⁸ is cycloalkyl of 3-6 carbon atoms which may be substituted as specified above in the definitions of R² and R³ ;

within these sub-groups, the following specific compounds are included:

II A: Z-2-isovaleramido-2-pentenoic acid; methyl Z-2-isovaleramido-2-butenoate; Z-2-isovaleramido-2-butenoic acid; Z-2-benzamido-2-butenoic acid; Z-2-(3,5,5-trimethylhexanamido)-2-butenoic acid; Z-2-cyclobutanecarboxamido-2-butenoic acid; Z-2-cyclopropanecarboxamido-2-butenoic acid; Z-2-cyclopropanecarboxamido-2-pentenoic acid; Z-2-(3-methylvaleramido)-2-butenoic acid; Z-2-cycloheptanecarboxamido-2-butenoic acid; Z-2-nonanamido-2-butenoic acid; Z-2-cyclohexanecarboxamido-2-butenoic acid; Z-2-(4-methylvaleramido)-2-butenoic acid; Z-2-t-butylacetamido-2-butenoic acid; Z-2-octanamido-2-butenoic acid; Z-2-butyramido-2-butenoic acid; Z-2-valeramido-2-butenoic acid; Z-2-valeramido-2-pentenoic acid; Z-2-cyclopentanecarboxamido-2-butenoic acid; Z-2-(6-methylheptanamido)-2-butenoic acid; Z-2-hexanamido-2-butenoic acid; Z-2-(3,7-dimethyloctanamido)-2-butenoic acid; Z-2-(3,7-dimethyl-6-octenamido)-2-butenoic acid; Z-2-(5-chlorovaleramido)-2-butenoic acid; Z-2-(3-chlorobenzoylamido)-2-butenoic acid; Z-2-(2-chlorobenzamido)-2-butenoic acid; Z-2-nonanamido-2-butenoic acid; Z-2-(6-bromohexanamido)-2-butenoic acid; Z-2-(3,3-dimethylpropenamido)-2 -butenoic acid; Z-2-benzamido-2-cinnamic acid; Z-2-benzamido-2-pentenoic acid; Z-2-benzamido-5-methoxy-2-pentenoic acid; Z-2-benzamido-2-hexenedioic acid; Z-2-isovaleramido-2-octenoic acid; Z-2-isovaleramido-2-cinnamic acid; Z-2-isovaleramido-2-hexenedioic acid; Z-2-cyclopropanecarboxamido-2-cinnamic acid; Z-2-cyclopropanecarboxamido-2-hexenedioic acid; Z-2-(5-methoxy-3-methylvaleramido)-2-butenoic acid; Z-2-ethylthioacetamido-2-butenoic acid; Z-2-(2,2-dichlorocyclopropanecarboxamido)-2-butenoic acid; Z-2-(2-ethylhexanamido)-2-butenoic acid; Z-2-di-n-propylacetamido-2-butenoic acid;

II B: Z-2-(2,2-dimethylcyclopropanecarboxamido)-2-butenoic acid; (+)-Z-2-(2,2-dimethylcyclopropanecarboxamido)-2-butenoic acid; Z-2-(2,2-dimethylcyclopropanecarboxamido)-2-pentenoic acid; Z-2-(2,2-dimethylcyclopropanecarboxamido)-2-octenoic acid; Z-2-(2,2-dimethylcyclopropanecarboxamido)-2-hexenoic acid; Z-2-(2,2-dimethylcyclopropanecarboxamido)-2-cinnamic acid; Z-2-(2,2-dimethylcyclopropanecarboxamido)-5-methoxy-2-pentenoic acid; Z-2-(2,2-dimethylcyclopropanecarboxamido)-4,4,4-trifluoro-2-butenoic acid; Z-2-(2,2-dimethylcyclopropanecarboxamido)-3-(2-chlorophenyl)propenoic acid; Z-2-(2,2-dimethylcyclopropanecarboxamido)-2-hexenedioic acid; Z-2-(2-ethylcyclopropanecarboxamido)-2-butenoic acid; Z-2-(2,2-diethylcyclopropanecarboxamido)-2-butenoic acid; Z-2-(2,2-diethylcyclopropanecarboxamido)-2-pentenoic acid; Z-2-(2-isopropyl-2-methylcyclopropanecarboxamido)-2-butenoic acid; Z-2-(2-methylcyclohexanecarboxamido)-2-butenoic acid; Z-5-cyano-2-(2,2-dimethylcyclopropanecarboxamido)-2-pentenoic acid; Z-5-(N,N-dimethylcarbamoyl)-2-(2,2-dimethylcyclopropanecarboxamido)-2-pentenoic acid; Z-2-(2,2-dimethylcyclopropanecarboxamido)-5-methanesulfonyl-2-pentenoic acid; Z-2-(2,2-dimethylcyclopropanecarboxamido)-5-ethoxycarbonyl-2-pentenoic acid; Z-2-(2-methylcyclopropanecarboxamido)-2-butenoic acid; methyl Z-2-(2,2-dimethylcyclopropanecarboxamido)-2-butenoate; ethyl Z-2-(2,2-dimethylcyclopropanecarboxamido)-2-butenoate; 2-dimethylaminoethyl ester of Z-2-(2,2-dimethylcyclopropanecarboxamido)-2-butenoic acid; 3-diethylaminopropyl ester of Z-2-(2,2-dimethylcyclopropanecarboxamido)-2-pentenoic acid; Z-2-(2,3-dimethylcyclopropanecarboxamido)-2-butenoic acid; Z-2-(3,3-dimethylcyclobutanecarboxamido)-2-butenoic acid; Z-2-(2-spirocyclopentanecarboxamido)-2-butenoic acid; Z-2-(2-t-butyl-3,3-dimethylcyclopropanecarboxamido)-2-butenoic acid; Z-2-(2,2-dimethylcyclopropanecarboxamido)-4-methyl-2-pentenoic acid; Z-2-(2-t-butylcyclopropanecarboxamido)-2-butenoic acid; Z-2-(2-phenylcyclopropanecarboxamido)-2-butenoic acid; Z-3-cyclohexyl-2-(2,2-dimethylcyclopropanecarboxamido)propenoic acid; Z-5-carboxy-5-(2,2-dimethylcyclopropanecarboxamido)-4-pentenamidine; Z-5-dimethyl amino-2-(2,2-dimethylcyclopropanecarboxamido)-2-pentenoic acid; Z-3-cyclopropyl-2-(2,2-dimethylcyclopropanecarboxamido)propenoic acid; Z-2-(2,2-dimethylcyclopropanecarboxamido)-2,5-hexadienoic acid; Z-2-(2,2-dimethylcyclopropanecarboxamido)-4-phenyl-2-butenoic acid; Z-2-(2,2-dimethylcyclopropanecarboxamido)-6-mercapto-2-hexenoic acid; Z-2-(2,2-dimethylcyclopropanecarboxamido)-5-methylthio-2-pentenoic acid; Z-2-(2,2-dimethylcyclopropanecarboxamido)-5-phosphono-2-pentenoic acid; Z-2-(2,2-dimethylcyclopropanecarboxamido)-2-heptenoic acid; Z-2-(2,2 -dimethylcyclopropanecarboxamido)-5-phenyl-2-pentenoic acid; Z-2-(2,2-dimethylcyclopropanecarboxamido)-2-nonenoic acid; Z-2-(2,2-dimethylcyclopropanecarboxamido)-2-decenoic acid; Z-2-(2,2-dimethylcyclopropanecarboxamido)-2-tridecenoic acid; Z-2-(2,2-dimethylcyclopropanecarboxamido)-6-methoxy-2-hexenoic acid (and 5-methoxy-2-pentenoic acid); Z-2-(2,2-dimethylcyclopropanecarboxamido)-6-methyl-2-heptenoic acid; Z-4-cyclohexyl-2-(2,2-dimethylcyclopropanecarboxamido)-2-butenoic acid;

II C: Z-2-cyclobutylacetamido-2-butenoic acid; Z-2-cyclopentylacetamido-2-butenoic acid; Z-2-cyclohexylacetamido-2-butenoic acid; Z-2-(4-cyclohexylbutyramido)-2-butenoic acid; Z-2-cyclopropylacetamido-2-butenoic acid; Z-2-cyclopropylacetamido-2-pentenoic acid; Z-2-(3-cyclopentylpropionamido)-2-butenoic acid; Z-2-(3-cyclohexylpropionamido)-2-butenoic acid; Z-2-(4-(2-thienyl)-butyramido)-2-butenoic acid; Z-2-(4-phenylbutyramido)-2-butenoic (D,L-α-lipoamido)-2-pentenoic acid; Z-2-(D,L-α-lipoamido)-2-cinnamic acid; Z-2-(3-(2-tetrahydrofuryl)-propionamido)-2-butenoic acid.

Particularly preferred substituents within the definition of R² above include the 2,2-dimethylcyclopropyl and the 2,2-dichlorocyclopropyl groups.

Within the definition of R³, particularly preferred groups of compounds include n-alkyl (1-9 carbons) and n-alkyl (1-9 carbons) having a terminal substituent which is a quaternary nitrogen, amine derivative, or amino acid derived group.

By the term "quaternary nitrogen" is meant a tetrasubstituted or heteroaromatic nitrogen which is positively charged. An ammonium moiety, substituted with hydrocarbon groups having 1-7 carbon atoms, which can be the same or different, is signified.

By the term "amino derivative" is meant a group such as amino, acylamino, ureido, amidino, guanidino and alkyl (1-7 carbon atoms) derivatives thereof.

By the term "amino acid derived group" is meant a moiety such as cysteinyl (--SCH₂ CH(NH₂)COOH) or sarcosyl (--N(CH₃)CH₂ COOH) in which a hydrogen joined to O, N or S of known amino acids is replaced.

Particularly preferred compounds from the most preferred groups of substituents of R² and R are those wherein R² is 2,2-dimethylcyclopropyl or 2,2-dichlorocyclopropyl, and R³ is a hydrocarbon chain of 3 to 7 carbon atoms without a terminal substituent, or having a terminal substituent which is trimethylammonium, amidino, guanidino, or 2-amino-2-carboxyethylthio. Names of specific examples of these include:

Z-2-(2,2-dimethylcyclopropanecarboxamido)-8-trimethylammonium hydroxide-2-octenoic acid inner salt;

Z-2-(2,2-dichlorocyclopropanecarboxamido)-8-trimethylammonium hydroxide-2-octenoic acid inner salt;

Z-2-(2,2-dimethylcyclopropanecarboxamido)-8-formamidino-2-octenoic acid;

Z-2-(2,2-dimethylcyclopropanecarboxamido)-8-guanidino-2-octenoic acid;

Z-2-(2,2-dimethylcyclopropanecarboxamido)-8-ureido-2-octenoic acid;

Z-8-(L-2-amino-2-carboxyethylthio)-2-(2,2-dimethylcyclopropanecarboxamido)-2-octenoic acid;

Z-2-(2,2-dimethylcyclopropanecarboxamido)-2-octenoic acid (racemic and dextrorotatory forms);

Z-2-(2,2-dichlorocyclopropanecarboxamido)-2-octenoic acid;

7-(L-2-amino-2-carboxyethylthio)-2-(2,2-dimethylcyclopropanecarboxamido)-2-heptenoic acid; and 6-(L-2-amino-2-carboxyethylthio)-2-(2,2-dimethylcyclopropanecarboxamido)-2-hexenoic acid.

The Z configuration (J. E. Blackwood et al., J. Am. Chem. Soc., 90, p. 509 (1968)) is assigned to the above compounds on the basis of their NMR spectra by analogy with the work of A. Srinavasan et al. [Tetrahedron Lett., 891 (1976)].

Although these compounds of Formula II, when R¹ is H, are described and named as the free acids, it will be apparent to one skilled in the art that various pharmaceutically acceptable derivatives such as alkali and alkaline earth metal, ammonium, or amine salts, or the like can be employed as equivalents thereto. Salts such as the sodium, potassium, calcium, or tetramethylammonium salts are suitable.

Some of the compounds of Formula II are novel compounds which are claimed in a copending U.S. application, U.S. Ser. No. 927,212, filed on July 24, 1978, now abandoned, and in U.S. Ser. No. 050,233, filed June 22, 1979, now abandoned; and in U.S. Ser. No. 188,178, filed Sept. 17, 1980, and which do not form part of this invention.

As mentioned above, the thienamycin-type compound is used in combination with the dipeptidase inhibitor.

METHODS OF TESTING AND USING THE INVENTION

Disposition studies with thienamycin, its natural analogs and its semi-synthetic derivatives have revealed a major metabolic degradation pathway of elimination in the various species examined (mouse, rat, dog, chimpanzee, Rhesus monkey). The extent of metabolism is reflected in low urinary recovery and short plasma half-lives. The nature of this degradation was demonstrated to be lactam cleavage by the renal dipeptidase (E.C.3.4.13.11), described first by Bergmann, M. and Schleich, H., Z. Physiol. Chem., 205 65 (1932); see also Greenstein, J. P., Advances in Enzymology, Vol. VIII, Wiley-Interscience, (1948), New York, and Campbell, B. J.; Lin, Y-C., Davis, R. V. and Ballew, E., "The Purification and Properties of Particulate Renal Dipeptidase", Biochim. Biophys. Acta., 118, 371 (1966).

In order to demonstrate the ability of the compounds of Formula II to suppress the action of the renal dipeptidase enzyme, an in vitro screen procedure was followed. This measured the ability of compounds to inhibit hydrolysis of glycyldehydrophenylalanine (GDP) by a solubilized preparation of dipeptidase isolated from hog kidneys. The procedure is as follows: to a 1 ml. system containing 50 mM "MOPS" (3-(N-morpholino)propanesulfonic acid) buffer, pH 7.1, is added 5 μg of lyophilized enzyme, and the test compound at a final concentration of 0.1 mM. After a five minute incubation at 37° C., GDP is added to a final concentration of 0.05 mM. Incubation is continued for 10 minutes, at 37° C. and hydrolysis of GDP is measured by the change in optical density with time at 275 nm. Inhibition of the enzyme is gauged by comparison to a standard run containing no inhibitor and is expressed as the inhibitor binding constant, K_(i). This is the concentration of the inhibitor which achieves 50% inhibition of enzyme.

The substrate GDP is employed in preference to thienamycin in this screen because it has a much higher maximal velocity of hydrolysis by renal dipeptidase, thereby reducing the amount of enzyme required. Both GDP and thienamycin have a similar affinity for renal dipeptidase; furthermore, K_(i) 's of inhibitors tested have been identical for the two substrates.

In addition to this in vitro screen procedure, an in vivo screen was followed to measure the test compound's ability to inhibit metabolism as reflected by increase in urinary recovery of the penems of formula I from the mouse. The procedure involves co-administration of the test compound by the intravenous or subcutaneous route at a dose-rate of 10-100 mg/kg, with 10 mg/kg penem. Penem recovery in the urine over a 4 hour period is then compared with its recovery in a control group to which test compound was not co-administered.

Urinary recovery of penem was measured in all cases with the use of a cylinder or disc diffusion assay, conducted in a manner described in U.S. Pat. No. 3,950,357. This bioassay, with Staphylococcus aureus ATCC 6538 as the test organism, has a useful response range from 0.04 μg/ml to 3.0 μg/ml.

The combination of the inhibitor and the penem can be in the form of a pharmaceutical composition containing the two compounds in a pharmaceutically acceptable carrier. The two can be employed in amounts so that the weight ratio of the penem to inhibitor is 1:3 to 30:1, and preferably 1:1 to 5:1.

The components can also be separately administered. For instance, the penem can be administered orally, intramuscularly or intravenously in amounts of 1-100 mg/kg/day, preferably 1-20 mg/kg/day, or 1-5 mg/kg/dose, in divided dosage forms, e.g., three or four times a day. The inhibitor can be separately administered, orally, intramuscularly, or IV, in amounts of 1-100 mg/kg/day, or preferably 1-30 mg/kg/day, or 1-5 mg/kg/dose in divided dosage forms, e.g., three or four times a day. The amounts of the two components administered during one day ideally are within the ratio limits denoted above.

One preferred dosage regimen and level is the combination of the compound 2-ethylthio-6-(1-hydroxyethyl)-pen-2-em-3-sodium carboxylate and the crystalline form of 7-(L-amino-2-carboxyethylthio)-2-(2,2-dimethylcyclopropanecarboxamido)-2-heptenoic acid, co-administered in a sterile aqueous IV injection form (sodium salt), at a level of 250 or 500 mg of the penem compound and about 1:1 (weight) of the heptenoic acid, or 250 or 500 mg. This dose can be given to humans (each assumed to weigh about 80 kg.) from 1 to 4 times daily, that is 3.1-25 mg/kg/day of each drug. Since this penem is orally active, it can also be combined with another inhibitor, ±Z-2-(2,2-dimethylcyclopropanecarboxamido)-2-octenoic acid and both administered orally, at dose levels (estimated for humans) at 2-8 mg/kg/dose of the penem and 1-8 mg/kg/dose of the inhibitor, such doses being administered 1-4 times a day.

The components, whether administered separately or together are employed in pharmaceutically acceptable carriers such as conventional vehicles adapted for oral adminstration such as capsules, tablets, or liquid solutions or suspensions. The components separately or together, can also be dissolved in a vehicle adapted for administration by injection. Suitable formulations for oral use, may include diluents, granulating agents, preservatives, binders, flavoring agents, and coating agents. The example of an oral use composition in the combination of active ingredients, or the acid component alone, intermixed in the dry pulverulent state with gelatin, starch, magnesium stearate, and alginic acid, and pressed into a tablet.

Examples which illustrate this invention follow.

SECTION 1. EXAMPLES ILLUSTRATING ACTIVITY Example 1

In Vitro Test Data

A 1 ml. system of 50 mM "MOPS" buffer, pH 7.1, is used. To this is added 5 μg of the pig renal enzyme and an amount of the test compound to bring its final concentration to 0.1 mM. After a five minute incubation at 37° C., an amount of GDP is added to bring its final concentration to 0.05 mM. The system is again incubated for 10 minutes, at 37° C. Hydrolysis of GDP is measured by its change in optical density with time at 275 nm. Inhibition of the enzyme is gauged by comparison to a standard run containing no inhibitor and is presented as percent inhibition. The K_(i) is a constant indicating the concentration of inhibitor necessary to produce 50% inhibition of enzyme. It is a calculated value obtained from running multiple in vitro assays, as above, at concentrations resulting in inhibition below and above the 50% inhibition point. The results are presented in Table I.

                                      TABLE I                                      __________________________________________________________________________      ##STR3##                                                                      Dipeptidase                             % Inhibition                                                                          K.sub.i                         Inhibitor                                                                            R.sup.3          R.sup.2          at 10.sup.-4 M                                                                        (μM)                         __________________________________________________________________________     1     CH.sub.2 CH.sub.3                                                                                ##STR4##        98     0.18                            2*    CH.sub.3                                                                                         ##STR5##        98     0.39                            2a*   CH.sub.3                                                                                         ##STR6##        100    0.12                            2b*   CH.sub.3                                                                                         ##STR7##               19.8                            3     CH.sub.3                                                                                         ##STR8##        92     1.7                             4     CH.sub.2 CH.sub.3                                                                                ##STR9##        87     3.2                             5     CH.sub.3                                                                                         ##STR10##       81     4.4                             6     CH.sub.3                                                                                         ##STR11##       83     4.6                             7     CH.sub.3                                                                                         ##STR12##       91     6                               8     CH.sub.3                                                                                         ##STR13##       80     6.2                             9     CH.sub.3                                                                                         ##STR14##       83     6.6                             10    CH.sub.3                                                                                         ##STR15##       97     9                               11    CH.sub.3                                                                                         ##STR16##       82     10                              12    (CH.sub.2).sub.4 CH.sub.2                                                                        ##STR17##              0.055                           13    (CH.sub.2).sub.5 N.sup.+ (CH.sub.3).sub.3                                                        ##STR18##              0.18                            14    (CH.sub.2).sub.5 N.sup.+ (CH.sub.3).sub.3                                                        ##STR19##              1.11                            15                                                                                    ##STR20##                                                                                       ##STR21##              0.72                            16                                                                                    ##STR22##                                                                                       ##STR23##              0.89                            17                                                                                    ##STR24##                                                                                       ##STR25##              0.21                            18    CH.sub.3         CH.sub.2 C(CH.sub.3).sub.3                                                                      75     20                              19    CH.sub.3         (CH.sub.2).sub.6 CH.sub.3                                                                       72     26                              20    CH.sub.3         (CH.sub.2).sub.2 CH.sub.3                                                                       69     30                              21    CH.sub.3                                                                                         ##STR26##       68     30                              22    CH.sub.3                                                                                         ##STR27##       64     22                              23    CH.sub.3         (CH.sub.2).sub.3 CH.sub.3                                                                       64     32                              24    CH.sub.3                                                                                         ##STR28##       59     30                              25    CH.sub.3         (CH.sub.2).sub.4 CH(CH.sub.3).sub.2                                                             57                                     26    CH.sub.3                                                                                         ##STR29##       56                                     27    CH.sub.3                                                                                         ##STR30##       54                                     28    CH.sub.3         CH.sub.2(CH.sub.2).sub.3 CH.sub.3                                                               54     39                              29    CH.sub.3         (CH.sub.2).sub.5 CH.sub.3                                                                       49                                     30    CH.sub.3         CH(CH.sub.2 CH.sub.3)CH.sub.2 CH.sub.2 CH.sub.2                                CH.sub.3         33                                     31    CH.sub.3         CH(CH.sub.2 CH.sub.2 CH.sub.3).sub.2                                                            13                                     32    CH.sub.3         CH(CH.sub.3).sub.2                                                                              31                                     33    HOOCH.sub.2 CH.sub.2                                                                             ##STR31##       90     5                               34    CH.sub.3                                                                                         ##STR32##       88     9                               35    CH.sub.3         CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2                                                    70     19                              36    CH.sub.3         CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 Cl                                                          64     20                              37    CH.sub.3                                                                                         ##STR33##       72     11                              38    CH.sub.3                                                                                         ##STR34##       90     6.5                             39    CH.sub.3 (CH.sub.2).sub.4                                                                       CH.sub.2CH(CH.sub.3 ).sub.2                                                                     95     2.6                             40    CH.sub.3                                                                                         ##STR35##       100    0.45                            41    (CH.sub.3).sub.2 CH                                                                              ##STR36##       98     0.54                            42    CH.sub.3                                                                                         ##STR37##       98     0.86                            43    CH.sub.3                                                                                         ##STR38##       96     1.6                             44    CH.sub.3                                                                                         ##STR39##       95     3                               45    CH.sub.3 CH.sub.2                                                                                ##STR40##       98     0.18                            46    Ph                                                                                               ##STR41##       100    0.62                            47    CH.sub.3 CH.sub.2 CH.sub.2                                                                       ##STR42##       98     0.11                            48                                                                                    ##STR43##                                                                                       ##STR44##       97     0.23                            49    CH.sub.3 (CH.sub.2).sub.3                                                                        ##STR45##       100    0.11                            50    CH.sub.3 (CH.sub.2).sub.4                                                                        ##STR46##       100    0.17                            51    HOOCCH.sub.2 CH.sub.2                                                                            ##STR47##       98     0.145                           52                                                                                    ##STR48##                                                                                       ##STR49##       100    0.15                            53    PhCH.sub.2 CH.sub.2                                                                              ##STR50##       96     0.33                            54    CH.sub.3 SCH.sub.2 CH.sub.2                                                                      ##STR51##       99     0.12                            55    CH.sub.3 SO.sub.2 CH.sub.2 CH.sub.2                                                              ##STR52##       96     0.5                             56    CH.sub.3 (CH.sub.2).sub.5                                                                        ##STR53##       98     0.149                           57    CH.sub.3 (CH.sub.2).sub.6                                                                        ##STR54##       99     0.092                           58    CH.sub.3 (CH.sub.2).sub.9                                                                        ##STR55##       96     0.14                            59    PhCH.sub.2                                                                                       ##STR56##       98     0.44                            60    CH.sub.3 O(CH.sub.2).sub.3                                                                       ##STR57##              0.28                            61    CH.sub.3 OCH.sub.2 CH.sub.2                                                                      ##STR58##       98     0.32                            62    (CH.sub.3).sub.3 CCH.sub.2                                                                       ##STR59##              0.34                            63    (CH.sub.3).sub.2 CHCH.sub.2 CH.sub.2                                                             ##STR60##       98     0.15                            64    H.sub.2 OC(CH.sub.2).sub.3                                                                       ##STR61##       99     0.048                           65                                                                                    ##STR62##                                                                                       ##STR63##              0.39                            66    CH.sub.3 (CH.sub.2).sub.4                                                                        ##STR64##              .08                             __________________________________________________________________________      *Compounds 2, 2a, and 2b are the racemic, dextrorotatory and levorotatory      forms respectively.                                                      

Example 1

An in vivo assay is conducted using 34 Charles River CD female mice. Seventeen of the mice are administered 40 mg/kg of the inhibitor 7-(L-2-amino-2-carboxyethylthio)-2-(2,2-dimethylcyclopropanecarboxamido)-2-heptenoic acid subcutaneously, and an additional 40 mg/kg per os. Immediately thereafter they and an additional 17 mice are administered 10 mg/kg of the thienamycin class compound (±)-[5R,6S]-2-ethylthio-6[(R)-1-hydroxyethyl]-pen-2-em-3-sodiocarboxylate by oral gavage. The level of the latter compound in plasma is tested at 10, 30, 60 and 90 minutes using 3 mice from each group at each time period. The following data are obtained.

    ______________________________________                                         Time       Plasma Level (m/ml)                                                                           Penem                                                (minutes)  Penem Alone    Plus Inhibitor                                       ______________________________________                                         10         3.1            2.6                                                  30         3.7            4.8                                                  60         4.4            6.1                                                  90         1.8            3.8                                                  ______________________________________                                    

The remaining 5 mice from each group are tested for urinary recovery of the penem with the following results.

    ______________________________________                                         Time         Penem                                                             After        +        % Urinary Recovery                                       Administration                                                                              Inhibitor                                                                               Penem Alone                                              ______________________________________                                         1 hour       1.5%     1.8%                                                     3 hours      2.3%     0.6%                                                     Total        3.8%     2.4%                                                     ______________________________________                                    

Example 2

A single animal crossover study in the chimpanzee which shows increased urinary recovery was conducted as follows: A male chimpanzee, body weight 60 kg, was given 5 mg/kg of the compound 2-ethylthio-6-(1-hydroxyethyl)-pen-2-em-3-sodio carboxylate, IV, alone, and in a second trial in combination with 5 mg/kg 7-(L-2-amino-2-carboxyethylthio)-2-(2,2-dimethylcyclopropanecarboxamido)-2-heptenoic acid. Urinary recovery of the penem was 21% when the penem was used alone, and this recovery rate rose to 77% when the combination was administered.

SECTION 2. EXAMPLES ILLUSTRATING CHEMICAL PREPARATIONS

The inhibitor compounds are novel compounds claimed in a copending application. These compounds are made by condensing directly the appropriate 2-keto acid and amide: ##STR65## wherein R² and R³ are as defined. The general reaction conditions involve mixing approximately 1-4:1 parts of the acid to the amide in an inert solvent such as toluene or methyl isovalerate and heating at reflux with azeotropic removal of water for from 3-48 hours, preferably 5-24 hours. The solution when cooled normally yields the product in crystalline form, but the product can also be isolated using a base extraction process. The product can be recrystallized by using generally known techniques. An optional modification of this procedure requires an additional small amount of p-toluenesulfonic acid as catalyst during the reaction.

Another route to the novel inhibitor compounds uses an α-amino acid, t-butyl ester in reaction with an acid chloride: ##STR66## This reaction takes place in the presence of base, such as triethylamine, in a solvent such as methylene chloride. The resulting N-acylated product (VII) is then oxidized by treatment with t-butyl hypochlorite followed by addition of sodium methoxide. This yields the 2-methoxy derivative (VIII) and/or its elimination product, the α,β-unsaturated ester (IX). Further treatment with anhydrous hydrochloric acid converts either VIII or IX (or the mixture of both) to the desired α,β-unsaturated free acid (II). ##STR67##

Some compounds wherein R³ has a terminal substituent which is an amino, quaternary nitrogen, thiol or carboxyl, derivative can be made most conveniently from an intermediate having a terminal bromine. In this case the intermediate has the structure ##STR68## wherein n is the number of carbons in the desired hydrocarbon chain (e.g., from 3-7).

In order to prepare R³ having a terminal trimethylammonium substituent, the bromo intermediate is reacted with trimethylamine; to yield the amino, the bromo intermediate is reacted with ammonia; the guanidino, reaction is with guanidine; to prepare the thio derivatives, including 2-amino-2-carboxyethylthio, the bromo compound is reacted with cysteine HCl, or the appropriate mercaptan. Derivatized amino, such as formamidino, ureido, and acylamide (acetamido) can be made from the compounds having an amino group by reacting with o-benzyl formimidate HCl, potassium cyanate and the appropriate acyl anhydride (acetic anhydride), respectively.

Another route for preparing compounds when R³ is a terminally substituted thio derivative utilizes a chloro-keto ester intermediate: ##STR69## in reaction with the desired amide, ##STR70## in toluene at reflux in the presence of a catalytic amount of p-toluenesulfonic acid. The resulting intermediate is hydrolyzed to the acid; the chloro group is then displaced in reaction with the appropriate mercaptan. This reaction is valuable since it permits use of the chiral amide IV, thereby preparing a functionalized side chain. In addition, the mixture of Z+E isomers prepared after the mercaptan condensation can be directly isomerized into the Z form by adding acid to a pH about 3, and heating to about 90° C. for 30 minutes. Only the Z form remains, and recovery is simple and straight forward.

More detail about preparation of the compounds is found in the following examples.

Example 6 Z-2-Isovaleramido-2-butenoic Acid

A solution of 1.07 g (10.5 mmole) of 2-ketobutyric acid and 0.71 g (7.0 mmole) of isovaleramide in 15 ml of toluene was stirred under reflux with collection of H₂ O in a small Dean-Stark trap. After 5 hrs, the solution was cooled, resulting in fairly heavy crystallization. After standing, the solid was collected on a filter and washed with toluene and then with CH₂ Cl₂. Yield of white crystals=0.47 g, mp 172°-174° (slight prelim. softening). The material was recrystallized from diisopropyl ketone. Tlc (4:1 toluene-AcOH) now showed only a faint trace of the other isomer. Yield of white crystals=0.32 g (25%), mp 175° (slight prelim. softening). NMR indicated essentially exclusively Z-isomer.

    ______________________________________                                         Anal. (C.sub.9 H.sub.15 NO.sub.3)                                                                 Calcd.  Found                                               ______________________________________                                         C                  58.36   58.59                                               H                  8.16    8.55                                                N                  7.56    7.43                                                ______________________________________                                    

Example 7 Z-2-(2,2-Dimethylcyclopropanecarboxamido)-2-pentenoic acid

A solution of 1.74 g (15 mmole) of 2-ketovaleric acid and 1.13 g (10 mmole) of 2,2-dimethylcyclopropanecarboxamide in 20 ml of toluene was refluxed with stirring with collection of H₂ O in a small Dean-Stark trap. After 20 hrs. the solution was cooled and treated with a gentle stream of N₂. Before much of the solvent had evaporated, crystallization was induced by scratching. After standing, the solid was collected on a filter and washed with toluene and some Et₂ O. Yield of white crystals=0.63 g (30%), mp 154.5°-155.5° (slight prelim. softening). Tlc (4:1 toluene-AcOH) showed only an extremely faint trace of the other isomer. NMR was consistent with the Z-configuration.

    ______________________________________                                         Anal. (C.sub.11 H.sub.17 NO.sub.3)                                                                Calcd.  Found                                               ______________________________________                                         C                  62.53   62.86                                               H                  8.11    8.27                                                N                  6.63    6.75                                                ______________________________________                                    

Example 8 Z-2-(3-Cyclopentylpropionamido)-2-butenoic acid

A solution of 1.41 g (10 mmole) of 3-cyclopentylpropionamide and 1.53 g (15 mmole) of 2-ketobutyric acid was stirred and refluxed under a small Dean-Stark trap. After 8 hrs. the solution was cooled, resulting in heavy crystallization. The solid was collected on a filter and washed with toluene and CH₂ Cl₂. Yield of white crystals=1.44 g, mp 180.5°-182° (prelim. softening). The material was recrystallized from methyl ethyl ketone. Yield of white needles=0.63 g (28%), mp 184°-185° (slight prelim. softening). Tlc (4:1 toluene-AcOH) now showed a single spot, and NMR indicated essentially pure Z-isomer.

    ______________________________________                                         Anal. (C.sub.12 H.sub.19 NO.sub.3)                                                                Calcd.  Found                                               ______________________________________                                         C                  63.97   63.99                                               H                  8.50    8.67                                                N                  6.22    6.27                                                ______________________________________                                    

Example 9 Z-2-(2-Ethylhexanamido)-2-butenoic acid

10 g. of 2-ethylhexanoyl chloride was added dropwise with stirring to 25 ml of cold conc. NH₄ OH solution, resulting in immediate precipitation. The mixture was allowed to stir for 2 hrs., then filtered, and air dried to give 6.5 g. of amide. 1.4 g (10 mmole) of the above compound and 1.5 g of ketobutyric acid (15 mmole) were refluxed in 25 ml toluene for 15 hrs with removal of water. The reaction mixture was cooled and partly evaporated with a stream of N₂. Crystallization of product occurred after standing for 3 hrs. The crystals were collected, washed 3× with toluene, and air dried. There was isolated 1.13 g (50%) of product, mp 160°-162°. NMR was in accord with the assigned structure and indicated <5% E isomer. Tlc (4:1 toluene-AcOH) showed a single spot.

    ______________________________________                                         Anal. (C.sub.12 H.sub.21 NO.sub.3)                                                                Calcd.  Found                                               ______________________________________                                         C                  63.40   63.63                                               H                  9.30    9.43                                                N                  6.16    5.88                                                ______________________________________                                    

Example 10 Z-2-(2,2-Dimethylcyclopropanecarboxamido)-2-butenoic acid

1.53 g (15 mmoles) of 2-ketobutyric acid, 1.13 g (10 mmoles) of 2,2-dimethylcyclopropanecarboxamide and 20 ml of toluene stirred at reflux for 10 hours. After cooling the crystalline solid was filtered and washed with toluene (3×10 ml) and dried to give 1.06 g of product, mp 140°-141° C. Tlc (4:1 toluene-AcOH) showed essentially one spot and the NMR spectrum fit the desired structure.

Recrystallization from EtOAc gave after drying 0.533 g of product mp 142°-143.5°, homogeneous by tlc.

    ______________________________________                                         Anal. (C.sub.10 H.sub.15 NO.sub.3)                                                                Calcd.  Found                                               ______________________________________                                         C                  60.90   60.92                                               H                  7.67    7.71                                                N                  7.10    7.38                                                ______________________________________                                    

Example 11 Z-2-(2,2-Dimethylcyclopropanecarboxamido)-2-hexenedioic acid

A mixture of 1.0 g. of 2,2-dimethylcyclopropanecarboxamide, 2.4 g. of 2-ketoadipic acid and 25 ml. of methyl isovalerate was heated under reflux for 4 hrs, with removal of H₂ O by a modified Dean-Stark trap containing molecular sieves (4A). After standing at room temperature overnight, the crystalline precipitate was filtered, washed with ether and recrystallized from ethyl acetate to give 0.23 g. of product, m.p. 163°-165°. The NMR spectrum was consistent with the desired structure.

    ______________________________________                                         Anal. (C.sub.12 H.sub.17 NO.sub.5)                                                                Calcd.  Found                                               ______________________________________                                         C                  56.46   56.20                                               H                  6.71    6.83                                                N                  5.49    5.32                                                ______________________________________                                    

Example 12 Z-2-(2,2-Diethylcyclopropanecarboxamido)-2-butenoic acid

A mixture of 2.3 g of 2-ketobutyric acid, 2.0 g of 2,2-diethylcyclopropanecarboxamide, and 25 ml of toluene was heated under reflux for 16 hrs with removal of H₂ O by a modified Dean-Stark trap containing molecular sieves (4A). No product precipitated upon cooling. Ether (25 ml) was added and the mixture was extracted with saturated NaHCO₃ (3 times). The combined extracts were acidified with concentrated HCl. The gummy precipitate crystallized when triturated with water. Recrystallization from ethyl acetate gave 0.31 g of product, m.p. 129°-30°. The NMR spectrum was consistent with the desired structure.

    ______________________________________                                         Anal. (C.sub.12 H.sub.19 NO.sub.3)                                                                Calcd.  Found                                               ______________________________________                                         C                  63.98   64.01                                               H                  8.50    8.62                                                N                  6.22    6.21                                                ______________________________________                                    

Example 13 2-(2,2-Dimethylcyclopropanecarboxamido)-2-hexenoic acid Step A: DL-Norleucine t-butyl ester

General procedure of R. Roeske, J. Org. Chem. 28, p. 1251 (1963).

To a suspension of 9.82 g (75 mmole) of DL-norleucine in 80 ml of dioxane in a 500 ml. pressure bottle cooled in an ice bath was added slowly (with swirling) 8 ml of concentrated H₂ SO₄. The resulting mixture was cooled in a dry ice bath as 80 ml of liquid isobutylene was added. The mixture was allowed to warm to room temperature and shaken under autogenous pressure for˜23 hrs. After most of the isobutylene had been vented off, the slightly hazy solution was cooled in ice and then added to a cold mixture of 400 ml of 1N NaOH and 500 ml of Et₂ O. After shaking in a separate funnel, the layers were separated, and the aqueous fraction was washed with an additional 100 ml of Et₂). The Et₂ O solution was shaken with 150 ml of 0.5N HCl. The acidic aqueous fraction was treated with 2.5N NaOH until strongly basic and then shaken with 250 ml of Et₂ O. The Et₂ O solution was dried (MgSO₄), filtered, and concentrated on the rotovac. After prolonged pumping on high vacuum over a steam bath, final yield of clear, colorless residual oil=9.04 g (65%). NMR now showed only a trace of dioxane. TLC (9:1 CHCl₃ -MeOH) showed a single spot.

Step B: N-(2,2-Dimethylcyclopropanecarbonyl)-DL-norleucine t-butyl ester

To a solution of 8.98 g (48 mmole) of DL-norleucine t-butyl ester and 5.05 g (50 mmole) of triethylamine in 100 ml of CH₂ Cl₂ stirred in an ice bath under a drying tube was added dropwise (over a period of 75 min.) a solution of 6.39 g (48 mmole) of 2,2-dimethylcyclopropanecarbonyl chloride (M. Elliot and N. R. James, British Pat. No. 1,260,847 (1972)) in 50 ml of CH₂ Cl₂. Precipitation of Et₃ N HCl occurred during the addition, especially toward the end. As the ice gradually melted, the mixture was allowed to warm to room temperature. After 16 hrs, the mixture was shaken with 200 ml of 0.5N HCl. The CH₂ Cl₂ fraction was washed with an additional 200 ml of 0.5N HCl, then with 2×200 ml of 0.5N NaOH, and finally 200 ml of H₂ O. The CH₂ Cl₂ fraction was dried with MgSO₄, treated with charcoal, and filtered through Celite. The filtrate was concentrated on the rotovac (finally under high vacuum). Yield of light orange residual oil=11.93 g (88%). Tlc (2:1 hexane-EtOAc) showed a single spot. NMR and IR were in accord with the assigned structure. After standing for several days, the unused portion of this material crystallized: m.p. 52°→65°.

Step C: t-Butyl 2-(2,2-dimethylcyclopropanecarboxamido)-2-methoxyhexanoate

Based on procedure of H. Poisel and V. Schmidt, Chem. Ber., 108 p. 2547 (1975).

To a solution of 6.37 g (22.5 mmole) of N-(2,2-dimethylcyclopropanecarbonyl)-DL-norleucine t-butyl ester in 35 ml of Et₂ O stirred at room temperature under N₂ in the dark was added 2.69 ml (2.45 g, 22.5 mmole) of t-butyl hypochlorite. After 15 min., a solution of sodium methoxide prepared by dissolving 0.52 g (22.6 mmole) of sodium in 35 ml of MeOH was added. Stirring was continued at ambient temperature under N₂ in the dark. After 16.5 hrs., the precipitated NaCl was filtered off. The filtrate was diluted with Et₂ O and washed successively with 3×50 ml of 0.5N HCl, 50 ml of saturated Na₂ CO₃, and 2×50 ml of H₂ O. The Et₂ O phase was dried over MgSO₄ and filtered. The filtrate was concentrated on the rotovac. The pale, golden-yellow residual oil (6.45 g) was subjected to preparative high pressure liquid chromatography, resulting in the separation and isolation of 273 mg and 496 mg of the two diastereomers of t-butyl 2-(2,2-dimethylcyclopropanecarboxamido)-2-methoxyhexanoate (respective mp's 114°-118° and 124°-125.5°) as well as 1.97 g of a single isomer (apparently Z) of t-butyl 2-(2,2-dimethylcyclopropanecarboxamido)-2-hexenoate (colorless oil).

Step D: 2-(2,2-Dimethylcyclopropanecarboxamido)-2-hexenoic acid

A solution of 0.84 g (3.0 mmole) of t-butyl 2-(2,2-dimethylcyclopropanecarboxamido)-2-hexenoate in 10 ml of Et₂ O saturated with anhydrous HCl was allowed to stand at room temperature under a drying tube. After 17 hrs, the solution was evaporated, and the residual gum was dissolved in 10 ml of saturated NaHCO₃. This solution was washed with an additional 15 ml of 0.5N HCl, then dried (MgSO₄), filtered, and concentrated to give a viscous oil. The oil was crystallized from toluene. Yield of white crystals=0.32 g (47%), m.p. 119°-122°. TLC (4:1 toluene-AcOH) showed a single spot. NMR indicated essentially pure Z-isomer. (Note: Treatment of the methanol adduct, t-butyl 2-(2,2-dimethylcyclopropanecarboxamido)-2-methoxyhexenoate, with anhydrous HCl in Et₂ O under similar conditions gave the same product.)

Example 14 (+)-Z-2-(2,2-Dimethylcyclopropanecarbonylamino)-2-octenoic acid, sodium salt

The reagents, (+)-2,2-dimethylcyclopropanecarboxamide, 7.0 g.; 2-keto-octanoic acid ethyl ester, 14.7 g.; 50 mg. of p-toluene sulfonic acid; and 100 ml. of toluene was changed to a 250 ml. three-necked flask under a Dean Stark trap containing several molecular sieve pellets. The mixture was refluxed vigorously for 27 hours. The resultant light yellow solution was cooled and concentrated in vacuo, at a water bath temperature of 45° C., in the presence of water to help remove toluene. The gummy residue was suspended in 230 ml. of 2N NaOH and stirred at 30° C. for 3 hours; then the temperature was raised to 35° C. for an additional 21/2 hrs. until a clear solution formed. The solution was then cooled, 85 ml. methylene chloride added, and the pH adjusted to 8.5 using 4N HCl with stirring. The organic layer was separated and discarded. The aqueous layer (366 ml.) was assayed by liquid chromatography to contain 37.2 mg/ml; 87% Z isomer. Another 85 ml. portion of CH₂ Cl₂ was then added and pH adjusted to 4.5 with stirring. The organic layer was separated and the aqueous layer reextracted with 50 ml. of CH₂ Cl₂, with the pH again adjusted to 4.5. Combined organic extracts were dried over Na₂ SO₄, filtered, and concentrated to a gum. This residue was dissolved in 150 ml. isopropanol and 15 ml. water and the pH adjusted to 8.2 with 2N NaOH. The resulting solution was concentrated to an oily residue which was flushed with isopropanol until it turned to a crystalline solid, indicating that most water had been removed. It was crystallized from 120 ml. of isopropanol, (cooled in ice for 1 hour) filtered, and washed with 50 ml. cold isopropanol followed by copious amounts of acetone. It was dried at 60° C./0.1 mm/2 hours to yield 10.74 g (63.2%) crystalline material, having essentially a single peak in liquid chromatography, m.p. 241°-243° C.

The starting material, (+)-2,2-dimethylcyclopropanecarboxamide is most conveniently prepared by resolution of the D,L acid, followed by reaction with oxalyl chloride and then ammonia to give the resolved amide.

One way of making the starting material is as follows: 23.1 g. of D,L-2,2-dimethylcyclopropanecarboxylic acid was suspended in 33 ml H₂ O and the pH adjusted to 8.0, using 50% NaOH, about 10 ml. To this was added a solution of 38.4 g quinine in a mixture of 60 ml. methanol and 30 ml. H₂ O to which had been added about 8 ml of concentrated HCl in another 30 ml. H₂ O to give a pH of 7.1. (This was actually a solution of quinine hydrochloride.)

These solutions were added all at once, with stirring. The gummy crystalline material which formed was heated to give two clear layers and again stirred vigorously while cooling to give a crystalline product. This product was permitted to stand over two days at room temperature. It was then filtered, washed with 2×10 ml water, and 2×10 ml 50% methanol, and air dried with suction. The yield of crude quinine salt was 44.8 g (48.7% yield) monohydrate, m.p. 113°-116° C., having a [α]_(D) ²⁰ of -94.3°, C=1.0; CHCl₃. This material was recrystallized from acetone to yield 24.35 g, m.p. 127°-130° C. This purified quinine salt was converted to the acid by reaction with aqueous base and chloroform, followed by acid, to yield (96%) 3.9 g having [α]_(D) ²⁰ of +146.0°.

This acid was converted to the amide as follows: A charge of 30.5 g (+) acid was added over 5-10 minutes through a dropping funnel to chilled (10° C.) oxalyl chloride, 54 ml., containing 1 drop dimethylformamide. This was stirred overnight at ambient temperature. A clear solution was observed, which was added to 100 ml. methylene chloride to dilute. Excess oxalyl chloride was removed by concentrating and the mixture flushed twice with methylene chloride.

The resultant solution was diluted with an equal volume of methylene chloride, and added continuously through a dropping funnel to about 100 ml. anhydrous liquid ammonia which was diluted with 100 ml methylene chloride. A dry ice-acetone cooling bath was used during the addition. When all was added, the cooling bath was removed and the mixture stirred at room temperature for about 1/2 hour. The mixture was filtered, to remove precipitated ammonium chloride, and concentrated to dryness. The crude weight was 26.6 g. (88%). It was redissolved in excess hot ethyl acetate and filtered through a preheated sintered glass funnel to separate from trace NH₄ Cl. Excess ethyl acetate was atmospherically distilled off. When half the volume remained, 130 ml of heptane were added, and ethyl acetate was continued to be distilled off, until the boiling point started to rise (to near 80° C.; much of product had already crystallized out). Heat was removed, and the mixture let cool gradually to about 30° C., then cooled with an ice bath to 0°-5° C. for about 1/2 hour. The product was recovered as nice silvery-white crystalline flakes, washed with 3× ethyl acetate/hexane mixture, 1/1.5 and air dried to constant weight. It weighed 23.3 g (77.1% yield overall, 87.6% recovery from crude), m.p.=135°-138° C. (varies with rate of heating). Angle of rotation was determined by dissolving 0.0543 g in 10 ml chloroform, [α]_(D) ²⁰ =+100.9°.

Example 15 Z-2-(2,2-Dichlorocyclopropanecarboxamido)-2-butenoic acid Step A: 2,2-Dichlorocyclopropanecarboxamide

A 7.1 g sample of 2,2-dichlorocyclopropanecarbonyl chloride (U.S. Pat. No. 3,301,896, issued Jan. 31, 1967) was added dropwise to 75 ml of concentrated ammonium hydroxide with vigorous stirring. The temperature of the reaction mixture was maintained below 10° C. with an ice bath. The mixture was stirred in the ice bath for 30 min., then at room temperature for 1 hr. The aqueous ammonia was evaporated under reduced pressure (bath at 50° C.). The solid residue was extracted with hot ethyl acetate (3×30 ml). The extracts were boiled down to 40 ml and 20 ml of hexane was added. After cooling in ice, the solid was filtered, washed with ethyl acetate-hexane (1:1) and dried to give 2.7 g of 2,2-dichlorocyclopropanecarboxamide, m.p. 144°-146°. The NMR spectrum was in accord with the desired structure.

    ______________________________________                                         Anal. (C.sub.4 H.sub.5 Cl.sub.2 NO)                                                               Calcd.  Found                                               ______________________________________                                         C                  31.20   31.26                                               H                   3.27    3.31                                               N                   9.10    9.11                                               Cl                 46.04   45.79                                               ______________________________________                                    

Another 1.3 g of amide, m.p. 143°-145° could be recovered from the mother liquor.

Step B: Z-2-(2,2-Dichlorocyclopropanecarboxamido)-2-butenoic acid

A mixture of 1.53 g (15 mmoles) of 2-ketobutyric acid, 1.54 g (10 mmoles) of 2,2-dichlorocyclopropanecarboxamide and 10 ml of toluene was heated under reflux for 12 hrs. with removal of H₂ O by a modified Dean-Stark trap containing molecular sieves (4A). An additional 0.7 g of 2-ketobutyric acid was added and the reaction mixture was heated under reflux for an additional 12 hrs. The mixture was cooled, diluted with 20 ml of toluene and extracted with saturated sodium bicarbonate (3×10 ml). The extracts were combined, washed with ether and acidified to pH 3 (pH meter) with concentrated hydrochloric acid. A gum precipitated which soon solidified. It was filtered, washed with water, dried and recrystallized from nitromethane to give 423 mg of Z-2-(2,2-dichlorocyclopropanecarboxamido)-2-butenoic acid, m.p. 188°-189.5° C. The NMR spectrum was in accord with the desired structure.

    ______________________________________                                         Anal. (C.sub.8 H.sub.9 Cl.sub.2 NO.sub.3)                                                         Calcd.  Found                                               ______________________________________                                         C                  40.36   40.48                                               H                   3.81    3.80                                               N                   5.88    5.91                                               Cl                 29.78   29.53                                               ______________________________________                                    

Example 16 Z-2-(2,2-Dichlorocyclopropanecarboxamido)-2-octenoic acid

A mixture of 1.19 g (7.5 mmoles) of 2-ketooctanoic acid, 0.77 g (5.0 mmoles) of 2,2-dichlorocyclopropanecarboxamide, and 5 ml toluene were reacted using the same procedure as in the previous example. The crude product (537 mg) was purified by conversion to the methyl ester (BF₃ /CH₃ OH), preparative TLC (silica gel G, 4:1 hexane-EtOAc) and saponification of the pure Z-methyl ester (0.3M LiOH/CH₃ OH) to give 88 mg of Z-2-(2,2-dichlorocyclopropanecarboxamido)-2-octenoic acid as a partially crystalline gum. NMR spectrum (DMSO-d₆): 9.68δ (s, 1H, NH), 6.50δ (t, 1H, ##STR71## 2.83δ (t, 1H, ##STR72## 1.97δ (d, 2H, ##STR73## 0.87δ (t, 3H, CH₃).

Example 17 Z-8-Bromo-2-(2,2-Dimethylcyclopropanecarboxamido)-2-octenoic acid

To a suspension of 14.4 g (0.3 mole) of 50% NaH dispersion in 360 ml of toluene cooled in an ice bath and in a N₂ atmosphere was added over 45 min. a solution of 146 g (0.6 moles) of 1,6-dibromohexane and 57.6 g (0.3 mole) of ethyl 1,3-dithiane-2-carboxylate in 120 ml of DMF. The cooling bath was removed and the mixture stirred at room temperature for 20 hrs. The reaction mixture was washed with water (3×210 ml), dried over MgSO₄ and evaporated under reduced pressure to give 179.5 g of a yellow oil containing the desired alkylated dithiane, 1,6-dibromohexane and mineral oil. This crude material was used in the next reaction without purification.

To a suspension of 426 g (2.4 moles) of N-bromosuccinamide in 800 ml of acetonitrile and 200 ml of H₂ O was added over 45 min. a solution of the crude dithiane in 100 ml of acetonitrile. The temperature of the reaction mixture was maintained below 25° C. with an ice bath. After stirring at 20° C. for 10 min. the dark red reaction mixture was poured into 2.1. of hexane-CH₂ Cl₂ (1:1). The solution was shaken with saturated NaHSO₃ (2×400 ml) and water (1×500 ml). Then 400 ml of saturated Na₂ CO₃ solution was added in small portions (vigorous CO₂ solution). After the foaming subsided the funnel was shaken and the aqueous phase separated. The organic layer was extracted with saturated Na₂ CO₃ solution (400 ml) and water (500 ml) and dried over MgSO₄. Removal of the solvent under reduced pressure gave 133.8 g of crude bromo ketoester containing 1,6-dibromohexane and mineral oil. This crude material was used in the next reaction without purification.

A mixture of 133.8 g of crude bromo ketoester, 133 ml of 50% hydrobromic acid and 267 ml of acetic acid was heated at 90° C. (internal temperature) for 75 min. The dark solution was evaporated under reduced pressure until most of the acetic acid was removed. The residue was dissolved in 500 ml of ether, washed with water (2×100 ml) and extracted with saturated NaHCO₃ (3×200 ml). The combined NaHCO₃ extracts were extracted with ether (2×100 ml) and acidified with concentrated HCl. The precipitated oil was extracted with ether (3×200 ml). The ether extracts were washed with water (1×100 ml) and saturated brine (1×100 ml) and dried over MgSO₄. Removal of the ether under reduced pressure gave 46.2 g of pure bromoketo acid. Homogeneous by TlC (silica gel, 4:1 toluene-acetic acid). The NMR spectrum was consistent with the desired product.

A mixture of 46.1 g (0.194 moles) of the bromoketo acid, 17.6 g (0.156 mole) of 2,2-dimethylcyclopropanecarboxamide and 450 ml of toluene was heated under reflux for 13 hrs., with collection of water in a small Dean-Stark trap. After cooling, the clear reaction mixture was extracted with saturated NaHCO₃ solution (4×100 ml). The combined extracts were washed with ether (2×100 ml) and then the pH was adjusted to 3.5 (pH meter) by addition of concentrated HCl. An oil precipitated which soon crystallized. The solid was filtered, washed well with water and dried. Recrystallization from acetonitrile gave 22.5 g of Z-8-bromo-2-(2,2-dimethylcyclopropanecarboxamido)-2-octenoic acid, m.p. 151°-153° C. Homogeneous by TLC (4:1 toluene-acetic acid). The NMR spectrum was consistent with the desired structure.

    ______________________________________                                         Anal. (C.sub.14 H.sub.22 BrNO.sub.3)                                                              Calcd   Found                                               ______________________________________                                         C                  50.61   50.66                                               H                   6.67    6.96                                               N                   4.22    4.45                                               Br                 24.05   23.95                                               ______________________________________                                    

The following ω-bromo compounds were prepared using the same procedure:

Z-6-Bromo-2-(2,2-dimethylcyclopropanecarboxamido)-2-hexenoic acid;

Z-7-Bromo-2-(2,2-dimethylcyclopropanecarboxamido)-2-heptenoic acid;

Z-9-Bromo-2-(2,2-dimethylcyclopropanecarboxamido)-2-2-nonenoic acid;

Z-10-Bromo-2-(2,2-dimethylcyclopropanecarboxamido)-2-decenoic acid;

Z-8-Bromo-2-(2,2-dichlorocyclopropanecarboxamido)-2-octenoic acid.

Example 18 Z-8-Dimethylamino-2-(2,2-dimethylcyclopropanecarboxamido)-2-octenoic acid

A solution of 664 mg (2 mmoles) of Z-8-bromo-2-(2,2-dimethylcyclopropanecarboxamido)-2-octenoic acid in 10 ml of 40% aqueous dimethylamine was allowed to stand at room temperature for 4 hrs. The solution was poured onto a 3.5×20 cm column of Dowex 50W-x8 (100-200 mesh, H⁺) ion exchange resin and the column eluted with water until the effluent was no longer acidic (˜200 ml). The column was then eluted with 300 ml of 2N ammonium hydroxide. The effluent was evaporated under reduced pressure to give 600 mg of a colorless glass. This material was dissolved in 3 ml of ethanol, filtered, and added dropwise to 200 ml of rapidly stirred acetone. A gummy solid precipitated which crystallized upon stirring for two days. The solid was filtered, washed with acetone, and dried to give 445 mg of Z-8-dimethylamino-2-(2,2-dimethylcyclopropanecarboxamido)-2-octenoic acid as colorless, hygroscopic crystals, m.p. 101°-112° C. Homogeneous by TLC (silica gel, in BuOH, HOAc, H₂ O, 4:1:1). NMR spectrum was consistent with desired structure.

    ______________________________________                                         Anal. (C.sub.16 H.sub.28 N.sub.2 O.sub.3.H.sub.2 O)                                                Calcd.  Found                                              ______________________________________                                         C                   61.12   61.03                                              H                   9.62    9.28                                               N                   8.91    8.67                                               ______________________________________                                    

The following ω-amino derivatives were prepared using essentially the same procedure.

Z-10-Dimethylamino-2-(2,2-dimethylcyclopropanecarboxamido)-2-decenoic acid;

Z-8-Amino-2-(2,2-dimethylcyclopropanecarboxamido)-2-octenoic acid;

Z-8-Dimethylamino-2-(2,2-dimethylcyclopropanecarboxamido)-2-octenoic acid;

Z-7-Dimethylamino-2-(2,2-dimethylcylclopropanecarboxamido)-2-heptenoic acid;

Z-2-(2,2-dimethylcyclopropanecarboxamido)-7-(N-methylpiperazinyl)-2-heptenoic acid;

Z-2-(2,2-dimethylcyclopropanecarboxamido)-8-pyrrolidino-2-octenoic acid;

Z-2-(2,2-dimethylcyclopropanecarboxamido)-8-(N-methylpiperazinyl)-2-octenoic acid;

Z-8-Allylamino-2-(2,2-dimethylcyclopropanecarboxamido)-2-octenoic acid;

Z-2-(2,2-dimethylcyclopropanecarboxamido)-8-piperidino-2-octenoic acid;

Z-2-(2,2-dimethycyclopropanecarboxamido)-8-propargylamino-2-octenoic acid;

Z-8-N-[1-Deoxy-(1-methylamino)-D-glucityl]-2-(2,2-dimethylcyclopropanecarboxamido)-2-octenoic acid;

Z-8-(1-Adamantylamino)-2-(2,2-dimethylcyclopropanecarboxamido-2-octenoic acid;

Z-8-Diallylamino-2-(2,2-dimethylcyclopropanecarboxamido-2-octenoic acid;

Z-8-(2,2-dimethylcyclopropanecarboxamido)-8-(2-hydroxyethylmethylamino)-2-octenoic acid;

Z-8-[(Carboxylmethyl)methylamino]-2-(2,2-dimethylcyclopropanecarboxamido)-2-octenoic acid;

Z-2-(2,2-dimethylcyclopropanecarboxamido)-8-diethylamino-2-octenoic acid;

Z-2-(2,2-dimethylcyclopropanecarboxamido)-8-[tris-(hydroxymethyl)methylamino]-2-octenoic acid;

Z-2-(2,2-dimethylcyclopropanecarboxamido)-10-(N-methylpiperazinyl)-2-decenoic acid;

Z-2-(2,2-dimethylcyclopropanecarboxamido)-8-[1-(phosphono)ethylamino]-2-octenoic acid;

Example 18A Z-8-[(Carboxymethyl)methylamino]-2-(2,2-dimethylcyclopropane carboxamido)-2-octenoic acid

3.32 g of Z-8-bromo-2-(2,2-dimethylcyclopropanecarboxamido)-2-octenoic acid, 1.0 g of CH₃ NH CH₂ CO₂ H, 3.5 g of Na₂ CO₃ and 30 ml of water were heated at 80° C. in N₂ for 1.5 hours. After purification, 1.0 g of product was prepared, calc. for C₁₇ H₂₈ N₂ O₅. 2H₂ O: C, 54.24; H, 8.57; N, 7.44; found C, 54.40; H, 8.34; N, 7.16.

Example 18B Z-2-(2,2-dimethylcyclopropanecarboxamido)-8[1-(phosphono)ethylamino]-2-octenoic acid

was prepared reacting the same bromo intermediate (335.1 mg) with 138.2 mg 1-aminoethane phosphoric acid, 435 mg Na₂ CO₃ in 5 ml water, following essentially the same procedure, Ki=0.16.

Example 19 Z-2-(2,2-Dimethylcyclopropanecarboxamido)-8-methylthio-2-octenoic acid

A stream of CH₃ SH gas was bubbled through a solution of 162 mg (3 mmoles) of sodium methoxide in 5 ml of methanol for 10 min. with cooling in an ice bath. The solution was allowed to warm to room temperature and 332 mg (1 mmole) of Z-8-bromo-2-(2,2-dimethylcyclopropanecarboxamido)-2-octenoic acid was added. The solution was heated under reflux for 30 min. in a N₂ atmosphere. Most of the methanol was evaporated under reduced pressure, the residue 2.5 N HCl. The precipitated oil was extracted with ether (3×). The ether extracts were washed with water, saturated brine and dried over MgSO₄. Removal of the ether under reduced pressure gave a colorless oil that crystallized upon standing. It was recrystallized from ether-hexane to give 178 mg of Z-2-(2,2-dimethylcyclopropanecarboxamido)-8-methylthio-2-octenoic acid, m.p. 82°-84° C. Homogeneous by TLC (toluene-acetic acid, 4:1). The NMR spectrum was in accord with the desired structure.

    ______________________________________                                         Anal. (C.sub.15 H.sub.25 NO.sub.3 S)                                                              Calcd.  Found                                               ______________________________________                                         C                  60.18   60.36                                               H                   8.42    8.68                                               N                   4.68    4.59                                               S                  10.69   10.87                                               ______________________________________                                    

The following compounds were prepared by similar methods.

Z-2-(2,2-dimethylcyclopropanecarboxamido)-8-ethoxythiocarbonylthio-2-octenoic acid;

Z-2-(2,2-dimethylcyclopropanecarboxamido)-8-(1-methyl-5-tetrazolylthio)-2-octenoic acid;

Z-2-(2,2-dimethylcyclopropanecarboxamido)-7-{[(methoxycarbonyl)methyl]thio}-2-heptenoic acid;

Z-8-Acetylthio-2-(2,2-dimethylcyclopropanecarboxamido)-2-octenoic acid;

Z-7-[(2-Amino-2-oxoethyl)thio]-2-(2,2-dimethylcyclopropanecarboxamido)-2-heptenoic acid;

6-(L-2-Amino-2-carboxyethylthio)-2-(2,2-dimethylcyclopropanecarboxamido)-2-hexenoic acid;

Z-8-(Carbomethoxymethylthio)-2-(2,2-dimethylcyclopropanecarboxamido)-2-ocetnoic acid;

Z-6-(Carbomethoxymethylthio)-2-(2,2-dimethylcyclopropanecarboxamido)-2-hexenoic acid;

Z-2-(2,2-dimethylcyclopropanecarboxamido)-6-(phosphonomethylthio)-2-hexenoic acid.

The compound 7-(L-amino-2-carboxyethylthio)-2-(2,2-dimethylcyclopropanecarboxamido)-2-heptenoic acid is prepared in a similar fashion as the above example, except that Z-7-bromo-2-(2,2-dimethylcyclopropanecarboxamido)-2-heptenoic acid (prepared as in Example 17) (185 mg, 1.05 mmoles) is dissolved in 2.02 ml NaOH solution (2.0N), and deoxygenated by bubbling a stream of nitrogen gas through it for a minute. Then cysteine.HCl (185 mg, 1.05 mmoles) is added all at once and the reaction stirred at room temperature in a N₂ atmosphere for 3 hours. The reaction mixture is applied to 2×20 cm column of Dowex 50×4 (100-200 mesh, H⁺), and eluted with 300 ml H₂ O, then 200 ml of 2N NH₃ solution. Ammonia evaporated under reduced pressure to give 284 mg of a yellowish glass. This product is dissolved in 4 ml ethanol, and the insoluble material filtered. The filtrate is added dropwise to rapidly stirred diethylether (150 ml). The solid which precipitates is filtered, washed with ether and dried to yield 171 mg product, having one spot (ninhydrin positive) in TLC (μBuOH, HOAc, H₂ O; 4:1:1) rf. about 6; NMR is consistent with the desired structure.

    ______________________________________                                         Anal. (C.sub.16 H.sub.26 N.sub.2 O.sub.5 S)                                                       Calcd.  Found                                               ______________________________________                                         C                  53.61   52.55                                               H                  7.31    7.40                                                N                  7.81    7.89                                                S                  8.94    9.63                                                ______________________________________                                    

Example 19A Sodium Z-7-(L-amino-2-Carboxyethylthio)-2-(2,2-dimethylcyclopropane carboxamido)-2-heptenoic acid A. Grignard preparation of Ethyl-7-Chloro-2-oxoheptanoate

Equimolar amounts (8 moles each) of 1-bromo-5-chloropentane and magnesium are reacted in tetrahydrofuran (960 ml) at 25° C. The flask is charged with the Mg in the THF and the bromochloropentane added over 1 hour, then aged 2 hours. After the reaction was judged complete, the reaction solution was added (cooled -15° C., to 16 moles of diethyloxalate in 1856 ml tetrahydrofuran, while maintaining the temperature at 10° C. 3N HCl was added to quench, keeping the temperature below 25° C. After stripping solvents, the calculated yield is 48.8% of the ethyl-1-chloro-6-oxoheptenoate.

B. Condensation and Hydrolysis

S-2,2-dimethylcyclopropyl carboxamide (1017 g), 2143.6 g of ethyl-7-chloro-2-ketoheptanoate, 9 liters of toluene and 12 g of p-toluene sulfonic acid were charged to a 22 L flask, and heated to reflux with stirring. After 23 hours, liquid chromatography showed the expected product ratio, and 4 L of toluene were removed under slightly reduced pressure. The pot was charged with water, neutralized to pH 7 with 2N NaOH, and vacuum distilled leaving a final pot volume of about 5 liters.

This was hydrolyzed by adding 1760 g of 50% aq. NaOH (4 liters water) and stirring overnight. The flask was charged with 4 L methylene chloride, and pH adjusted to 8.8 using HCl. Unreacted amide crystallized out. The organic layers were separated from water, and then evaporated. The gummy residue was dissolved in 8 L water containing 720 g, 50% NaOH, and to this solution was charged 1818 g L-cysteine HCl.H₂ O, 2 kg ice, 2484 g 50% NaOH and 1 L water.

The pH of this solution, after aging overnight at room temperature, is adjusted to 3.0 with conc. HCl, and the resulting gummy suspension heated to 95° C. to afford a clear solution. After 30 minutes, no E isomer could be detected by 1c. After work-up and purification, the overall yield was 2060 g, 87% yield. This material was recrystallized from acetonitrile. 1500 g of the recrystallized material was dissolved in 6 L water and 910 ml 3.88 N NaOH, then neutralized to pH 7, and lyophilized to afford 1569 g (98.6%) of the title compound; Analysis: Calcd., C, 50.52; H, 6.62; N, 7.36; S, 8.43; Na, 6.04; found, C. 50.71; H, 6.78; N, 7.49; S, 8.52; Na 5.92.

Example 19B

Z-8-[(2-Amino-2-oxoethyl)thio]-2-(2,2-dimethylcyclopropane carboxamido)-2-octenoic acid was also prepared in a similar manner, to that described in Example 19, above, using 3.3 gm of the bromo intermediate, 1.3 g of H₂ NC(═O)CH₂ SH, in 50 ml methanol. 1.6 gms of product, mp. 127°-128° C. was obtained.

Example 20 Z-2-(2,2-Dimethylcyclopropanecarboxamido)-8-trimethylammonium hydroxide-2-octenoic acid inner salt

A solution of 996 mg (3 mmoles) of Z-8-bromo-2-(2,2-dimethylcyclopropanecarboxamido)-2-octenoic acid in 15 ml of 25% aqueous trimethylamine was allowed to stand at room temperature for 3 hrs. The reaction mixture was poured onto a 2×25 cm column of IRA-410 (50-100 mesh, OH⁻) ion exchange resin and eluted with water until the effluent was no longer basic. The effluent was evaporated under reduced pressure to give 800 mg of a colorless glass. This material was dissolved in 20 ml of ethanol, filtered and diluted with 600 ml of acetone. After standing at room temperature overnight the crystalline solid which deposited was filtered, washed with acetone and dried to give 720 mg of Z-2-(2,2-dimethylcyclopropanecarboxamido)-8-trimethylammonium hydroxide-2-octenoic acid inner salt as hygroscopic crystals, m.p. 220°-222° C. Homogeneous by TLC (silica gel, in BuOH, HOAc, H₂ O, 4:1:1). NMR spectrum was consistent with desired structure.

    ______________________________________                                         Anal. (C.sub.17 H.sub.30 N.sub.2 O.sub.3)                                                         Calcd   Found                                               ______________________________________                                         C                  65.77   65.78                                               H                  9.74    9.98                                                N                  9.02    8.92                                                ______________________________________                                    

Other quaternary derivatives were prepared using essentially the same procedure; these are

Z-2-(2,2-Dimethylcyclopropanecarboxamido)-8-trimethylammonium hydroxide-2-octenoic acid inner salt;

Z-2-(2,2-Dimethylcyclopropanecarboxamido)-8-pyridinium hydroxide-2-octenoic acid inner salt;

Z-2-(2,2-Dimethylcyclopropanecarboxamido)-8-(2-hydroxyethyldimethylammonium hydroxide)-2-octenoic acid inner salt;

Z-2-(2,2-Dimethylcyclopropanecarboxamido)-10-trimethylammonium hydroxide-2-decenoic acid inner salt;

Z-8-(Benzyldimethylammonium hydroxide)-2-(2,2-dimethylcyclopropanecarboxamido)-2-octenoic acid inner salt;

Z-10-(Benzyldimethylammonium hydroxide)-2-(2,2-dimethylcyclopropanecarboxamido)-2-decenoic acid inner salt;

Z-2-(2,2-Dimethylcyclopropanecarboxamido)-9-trimethylammonium hydroxide-2-nonenoic acid inner salt;

Z-8-(2-Dimethylaminoethylammonium hydroxide)-2-(2,2-dimethylcyclopropanecarboxamido)-2-octenoic acid inner salt;

Z-2-(2,2-Dichlorocyclopropanecarboxamido)-8-trimethylammonium hydroxide-2-octenoic acid inner salt;

Example 21 Z-2-(2,2-Dimethylcyclopropanecarboxamido)-8-formamidino-2-octenoic acid

A 350 mg sample of Z-8-amino-2-(2,2-dimethylcyclopropanecarboxamido)-2-octenoic acid was dissolved in 10 ml of water and the pH adjusted to 8.5 with 2.5N NaOH. A total of 947 mg of benzyl formimidate hydrochloride was added at room temperature in small portions over 20 min. while the pH was maintained between 8-9 by addition of 2.5N NaOH. After stirring at room temperature for 30 min., the cloudy reaction mixture was extracted with ether (3×) and applied to a 2×2.5 cm column of an AG50W-X4 (Na⁺, 200-400 mesh) resin. After elution with water, the fractions containing the product were pooled and evaporated under reduced pressure. This material was dissolved in water and applied to a 2×25 cm column of an AGlX8 (HCO₃ ⁻, 200-400 mesh) resin. After elution with water, the fractions containing pure product were pooled and evaporated under reduced pressure. The residue was dissolved in a few ml of warm ethanol, filtered, and added dropwise to 200 ml of ether with rapid stirring. Filtration and washing with ether gave 243 mg of Z-2-(2,2-dimethylcyclopropanecarboxamido)-8-formamidino-2-octenoic acid as an amorphous solid. Homongeneous by TLC (n-BuOH, HOAc, H₂ O; 4:1:1). The NMR spectrum was in accord with the desired structure.

    ______________________________________                                         Anal. (C.sub.15 H.sub.25 N.sub.3 O.sub.3.1/3H.sub.2 O)                                             Calcd.  Found                                              ______________________________________                                         C                   59.69   60.04                                              H                    8.59    8.64                                              N                   13.92   13.57                                              ______________________________________                                    

The following amidino compounds were prepared using similar procedures:

Z-8-Acetamidino-2-(2,2-dimethylcyclopropanecarboxamido)-2-octenoic acid;

Z-8-N-Benzylformamidino-2-(2,2-dimethylcyclopropanecarboxamido)-2-octenoic acid;

Z-2-(2,2-Dimethylcyclopropanecarboxamido)-10-formamidino-2-decenoic acid;

Z-2-(2,2-Dimethylcyclopropanecarboxamido)-8-(2-imidazolinyl-amino)-2-octenoic acid.

Example 22 Z-2-(2,2-Dimethylcyclopropanecarboxamido)-8-guanidino-2-octenoic acid

To a solution of 2 mmoles of guanidine (prepared from 432 mg of guanidine sulfate and 630 mg of barium hydroxide octahydrate) in 7 ml of water was added 332 mg (1 mmole) of 8-bromo-2-(2,2-dimethylcyclopropanecarboxamido)-2-octenoic acid, and the solution was heated at 70° C. in a nitrogen atmosphere for 1 hr. The reaction mixture was applied to a 2×25 cm column of Dowex 50W-X8 (H⁺, 100-200 mesh). After elution with water the fractions containing the product were pooled and evaporated under reduced pressure. The residue was dissolved in several ml of warm ethanol and added dropwise to 100 ml of ether with rapid stirring. Filtration and washing with ether gave 107 mg of Z-2-(2,2-dimethylcyclopropanecarboxamido)-8-guanidino-2-octenoic acid as an amorphous electrostatic powder. Homogeneous by TLC (n-BuOH, HOAc, H₂ O; 4:1:1). NMR (D₂ O, NaOD): 6.48δ (t, 1H, ##STR74## 3.10δ (m, 2H, ##STR75## 2.10δ (m, 2H, ##STR76## 1.17δ (s, 3H, ##STR77## 1.12δ (s, 3H, ##STR78##

The following guanidino compound was prepared using the same procedure:

Z-2-(2,2-Dimethylcyclopropanecarboxamido)-8-(N,N-dimethylguanidino)-2-octenoic acid.

Example 23 Z-2-(2,2-Dimethylcyclopropanecarboxamido)-8-methoxy-2-octenoic acid

To a solution of 2.43 mmoles of sodium methoxide in 5 ml of methanol was added 332 mg (1 mmole) of 8-bromo-2-(2,2-dimethylcyclopropanecarboxamido)-2-octenoic acid. The solution was heated under reflux in a nitrogen atmosphere for 1 hr. The reaction mixture was evaporated under reduced pressure, the residue dissolved in water and acidified with 2.5N hydrochloric acid. The oil which precipitated was extracted with ether (3×). The ether extracts were washed with water, and saturated brine and dried over MgSO₄. Removal of the ether under reduced pressure gave a colorless oil that crystallized upon standing. It was recrystallized from ether-hexane to give 140 mg of Z-2-(2,2-dimethylcyclopropanecarboxamido)-8-methoxy-2-octenoic acid, m.p. 71°-72° C. Homogeneous by TLC (toluene-HOAc, 4:1). The NMR spectrum was in accord with the desired structure.

    ______________________________________                                         Anal. (C.sub.15 H.sub.25 NO.sub.4)                                                               Calcd.  Found                                                ______________________________________                                         C                 63.58   63.54                                                H                 8.89    9.12                                                 N                 4.94    5.16                                                 ______________________________________                                    

Using similar procedures, the following compounds were prepared:

Z-8-Cyano-2-(2,2-dimethylcyclopropanecarboxamido)-2-octenoic acid;

Z-7-Cyano-2-(2,2-dimethylcyclopropanecarboxamido)-2-heptenoic acid;

Z-9-Cyano-2-(2,2-dimethylcyclopropanecarboxamido)-2-nonenoic acid;

Z-2-(2,2-Dimethylcyclopropanecarboxamido)-7-sulfo-2-heptenoic acid sodium salt;

Z-2-(2,2-Dimethylcyclopropanecarboxamido)-8-sulfo-2-octenoic acid sodium salt;

Z-2-(2,2-Dimethylcyclopropanecarboxamido)-8-hydroxy-2-octenoic acid;

Z-8-Acetoxy-2-(2,2-dimethylcyclopropanecarboxamido)-2-octenoic acid.

The Z-8-cyano-2-(2,2-dimethylcyclopropanecarboxamido)-2-octenoic compound was prepared from 332 mg 8-bromo-2-(2,2-dimethylcyclopropanecarboxamido)-2-octenoic acid and 100 mg NaCN in 2 ml DMSO, heated at 80° C. for 30 minutes. After extraction and purification, 102 mg of a colorless solid, mp 99°-103° C. were recovered, analysis for C₁₅ H₂₂ N₂ O₃ : calcd., C, 64.73; H, 7.97; N, 10.06; found, C, 64.69; H, 8.14; N, 9.41. 

What is claimed is:
 1. An antibacterial composition comprising a combination of a penem having the following structure: ##STR79## wherein R is substituted or unsubstituted alkyl having 1 to 6 carbon atoms or --SR' wherein R' is substituted or unsubstituted:phenyl or phenyl alkyl having 7-12 carbon atoms, heterocyclyl or heterocyclylalkyl wherein alkyl has 1-3 carbon atoms and the heterocyclic moiety has 1-4 hetero atoms selected from O, N, or S; and the easily removable or pharmaceutically acceptable salt or ester derivatives thereof; and a dipeptidase (E.C.3.4.13.11) inhibitor compound of the following formula: ##STR80## wherein R² and R³ are hydrocarbon radicals in the range respectively of 3-10 and 1-15 carbon atoms; in these R² or R³ hydrocarbon chains 1-6 hydrogens may be replaced by halogens or a nonterminal methylene may be replaced by oxygen or sulfur, including oxidized forms of the latter; additionally, a terminal hydrogen in R³ can also be replaced by hydroxyl or thiol, which may be acylated or carbamoylated; or the hydrogen can be replaced by amino, which may be derivatized as in an acylamino, ureido, amidino, guanidino, or alkyl or substituted amino group, including guaternary nitrogen groupings; or, there may be replacement by carboxylic, phosphonic or sulfonic acid groups or esters or amides thereof, or cyano; or combinations thereof, such as a terminal amino acid grouping; and R¹ is hydrogen or lower alkyl (C₁₋₆) or dialkylaminoalkyl, or a pharmaceutically acceptable cation, and the weight ratio of the penem to the dipeptidase inhibitor being within the range of about 1:3 to about 30:1.
 2. The composition of claim 1 in which the combination is mixed with a pharmaceutical carrier.
 3. The composition of claim 2 in which the carrier is adapted for injection.
 4. The composition of claim 1 in which the penem is 2-ethylthio-6-(1-hydroxyethyl)-penem-2-em-3-carboxylic acid or sodium carboxylate.
 5. The composition of claim 1 in which the penem is 2-pyridylthio-6-(1-hydroxyethyl)-pen-2-em-3-carboxylic acid or sodium carboxylate.
 6. The composition of claim 1 in which the penem is 2-phenylthio-6-(1-hydroxyethyl)-pen-2-em-3-carboxylic acid or sodium carboxylate.
 7. The composition of claim 1 in which R² can be R⁴, wherein R⁴ is branched or cyclic hydrocarbon of 3-10 carbon atoms;--R⁵ R⁶, wherein R⁵ is cycloalkyl of 3-6 carbon atoms and R⁶ is either 1 or 2 alkyl substituents which may be joined to form another ring on the cycloalkyl group or R⁶ is 1 or 2 chloro substituents; or --R⁷ R⁸, wherein R⁷ is alkylene of 1-3 carbon atoms and R⁸ is cycloalkyl of 3-6 carbon atoms.
 8. The composition of claim 1 in which R² is straight, branched or cycloalkyl of 3-10 carbon atoms, providing the carbon adjacent to the carbonyl cannot be tertiary.
 9. The composition of claim 1 in which R² is

    --R.sup.5 R.sup.6

wherein R⁵ is cycloalkyl of 3-6 carbon atoms and R⁶ is either 1 or 2 alkyl substituents which may be joined to form another ring on the cycloalkyl group.
 10. The composition of claim 1 in which R² is

    --R.sup.7 R.sup.8

wherein R⁷ is an alkylene group of 1-3 carbon atoms and R⁸ is cycloalkyl of 3-6 carbon atoms.
 11. The composition of claim 1 in which R² is 2,2-dimethylcyclopropyl.
 12. The composition of claim 1 in which R² is 2,2-dichlorocyclopropyl.
 13. The composition of claim 1 in which the dipeptidase inhibitor is Z-2-isovaleramido-2-butenoic acid.
 14. The composition of claim 1 in which the dipeptidase inhibitor is Z-2-(2,2-dimethylcyclopropanecarboxamido)-2-butenoic acid.
 15. The composition of claim 1 in which the dipeptidase inhibitor is Z-2-(2,2-dimethylcyclopropanecarboxamido)-2-pentenoic acid.
 16. The composition of claim 1 in which the dipeptidase inhibitor is Z-2-(2,2-dimethylcyclopropanecarboxamido)-2-octenoic acid.
 17. The composition of claim 1 in which the dipeptidase inhibitor is Z-2-(2,2-dimethylcyclopropanecarboxamido)-2-heptenoic acid.
 18. The composition of claim 1 in which the dipeptidase inhibitor is the 2-dimethylaminoethyl ester of Z-2-(2,2-dimethylcyclopropanecarboxamido)-2-octenoic acid.
 19. The composition of claim 1 in which the dipeptidase inhibitor is Z-2-(2,2-dimethylcyclopropanecarboxamido)-8-[1-(phosphono)ethylamino]-2-octenoic acid.
 20. The composition of claim 1 in which the dipeptidase inhibitor is Z-2-(2,2-dimethylcyclopropanecarboxamido)-8-trimethylammonium-2-octenoic acid.
 21. The composition of claim 1 in which the dipeptidase inhibitor is Z-2-(2,2-dichlorocyclopropanecarboxamido)-8-trimethylammonium-2-octenoic acid.
 22. The composition of claim 1 in which the dipeptidase inhibitor is Z-2-(2,2-dimethylcyclopropanecarboxamido)-8-guanidino, or amidino, or ureido-2-octenoic acid.
 23. The composition of claim 1 in which the dipeptidase inhibitor is 6-(L-2-amino-2-carboxyethylthio)-2-(2,2-dimethylcyclopropanecarboxamido)-2-hexenoic acid.
 24. The composition of claim 1 in which the dipeptidase inhibitor is 7-(L-2-amino-2-carboxyethylthio)-2-(2,2-dimethylcyclopropanecarboxamido)-2-heptenoic acid.
 25. The composition of claim 1 in which one dipeptidase inhibitor is Z-8-[(carboxymethyl)methylamino]-2-(2,2-dimethylcyclopropane carboxamido)-2-octenoic acid.
 26. The composition of claim 1 in which the dipeptidase inhibitor is Z-8-[(2-amino-2-oxoethyl)thio]-2-[(2,2-dimethylcyclopropane carboxamido)-2-oxoethyl)thio]-2-(2,2-dimethylcyclopropane carboxamido)-2-octenoic acid.
 27. The composition of claim 1 in which the dipeptidase inhibitor is Z-8-cyano-2-(2,2-dimethylcyclopropane carboxamido)-2-octenoic acid.
 28. The composition of claim 1 in which the dipeptidase inhibitor is Z-8-acetamido-2-(2,2-dimethylcyclopropane carboxamido)-2-octenoic acid.
 29. The method of treating bacterial infections which comprises administering to an animal an antibacterially effective amount of a penem having the following structure: ##STR81## wherein R is substituted or unsubstituted alkyl having 1 to 6 carbon atoms, or --SR' wherein R' is substituted or unsubstituted: alkyl having 7-12 carbon atoms, heterocyclyl or heterocyclylalkyl wherein alkyl has 1-3 carbon atoms and the heterocyclic moiety has 1-4 hetero atoms selected from O, N, or S; and the easily removable or pharmaceutically acceptable salt or ester derivative thereof; and, either separately, or together, an amount of a dipeptidase (E.C.3.4.13.11) inhibitor of the following formula: ##STR82## wherein R¹, R² and R³ are as defined in claim 1, so that weight ratio of the penem to the inhibitor is within about 1:3 to about 30:1.
 30. The method of claim 29 in which the penem is 2-ethylthio-6-(1-hydroxyethyl)-pen-2-em-3-carboxylic acid or sodium carboxylate.
 31. The method of claim 29 in which the dipeptidase inhibitor is 7-(L-2-amino-2-carboxyethylthio)-2-(2,2-dimethylcyclopropane carboxamido)-2-heptenoic acid, or its sodium, potassium, calcium or magnesium salt form.
 32. The method of claim 29 in which the dipeptidase inhibitor is ±Z-2-(2,2-dimethylcyclopropanecarboxamido)-2-octenoic acid, or its sodium, potassium, calcium, or magnesium salt form. 